PB1817: THE ADDITION OF ZALCITABINE TO ACUTE MYELOID LEUKEMIA TREATMENT MAY IMPROVE AZACITIDINE AND VENETOCLAX COMBINATION
Authors: Álvarez-Galeano Sofía, Milton Salazar-Hidalgo, Celina Benavente, José Luis López-Lorenzo, Eduardo Anguita
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. It is a disease with a very high mortality (24% 5-years survival rate), especially in elderly patients, who are the most frequently affected. The basic treatment of AML has hardly changed in the last 50 years. Recently, demethylating and anti-BCL2 drugs have been introduced in elderly and unfit patients. However, these drugs have a low success rate and a limited period of effectiveness. Treatment of AML cells with 2′-3′-dideoxycytidine (ddC), also known as zalcitabine, has been shown to inhibit mitochondrial DNA replication and oxidative phosphorylation, leading to an anti-AML effect. Zalcitabine is a pyrimidine analog used for the treatment of HIV infection. Therapeutic experience against HIV has shown that it is a relatively non-toxic treatment. Aims: Our purpose is to establish the in vitro impact of the combination of zalcitabine (Z) with other drugs commonly used against AML in unfit patients: azacitidine (A) and/or venetoclax (V). Methods: Cell lines: We studied human AML OCIAML3 and MOLM13 lines, with different pattern of gene alterations to get a model closer the patient´s heterogeneity. OCIAML3 carries mutations of DNMT3A, NRAS and NPM1 and MOLM13 has KMT2A::MLLT3 rearrangement and FLT3ITD. Cells were cultivated in RPMI1640 Glutamax, with 100 U/ml of penicillin G, 100 μg/ml of streptomycin and supplemented with 10% (v/v) fetal bovine serum (FBS), in the presence of the drugs or their solvent (negative control). Drugs: Z was used in the published range (0.2-4 μM). The effective dose 50 (ED50) and 10 (ED10) of the individual drugs (V and A) were calculated for each cell line within the published ranges (0.01-25 μM). For combinations, we used the range of Z and the ED50 of one conventional drug (A or V) or the ED10 of both (A plus V). Flow cytometry: Apoptosis was studied with PE Annexin-V Apoptosis Detection Kit I (BD Biosciences). The analysis was performed with CytExpert software. Results: To explore whether zalcitabine (Z) can potentiate azacitidine (A) and venetoclax (V) treatment of LAM, we studied the combination of these drugs in vitro. First, we established the ED50 of A and V for each cell line analyzing the percentage of Annexin-V positive cells (total apoptotic cells) of the human AML cells lines OCIAML3, which was sensitive to Z in our previous assays and MOLM13, resistant to Z in our initial studies. The combination of the ED50 for each line of A, with the increasing range of concentrations of Z caused increased apoptosis, more than 20%, with the highest dose of Z tested (4 μM), compared to A in monotherapy, in both lines (Figure 1 A-B). In addition, we studied the combination of the ED50 of V with the aforementioned range of Z concentrations in both cell lines, observing approximately a 15% increase in the level of apoptosis with respect to V in monotherapy (Figure 1 C-D). Then, to test if Z could improve the azacitidine plus venetoclax combination, we determined the ED10 of each drug combined induced 50% cell death in both cell lines (A´+V´) (Figure 1 E-F). Again, A ED10, V ED10 and 4 μM Z (A´+V´+Z) significantly increased over a 20% the A´+V´ combination apoptosis level (Figure 1 E-F). In the figure, results are presented as mean ± SEM of 3 independent experiments. Significance levels were determined by one way ANOVA: *p < 0.05, **p < 0.01, ****p < 0.0001. C: control without drugs (DMSO vehicle only). Summary/Conclusion:In vitro analysis suggests that zalcitabine can improve azacitidine and venetoclax combination currently used as treatment for unfit AML patients.Keywords: Acute myeloid leukemia, Mitochondria, Venetoclax, Azacitidine